Lin28a protein functions as a positive regulator of cell proliferation by enhancing the translation of genes controlling cell growth and suppressing the maturation of let-7 microRNAs that promote cell differentiation. Accordingly, Lin28a gene products are highly expressed in pluripotent cells and certain cancer cells but are generally absent in differentiated cell types. We found that the Lin28a promoter DNA fragment was transcriptionally active in mouse cells that do not show endogenous Lin28a expression. The lack of correlation between promoter CpG hypermethylation and silencing of gene expression suggested that Lin28a transcription was not repressed by DNA methylation. Consistently, 5-aza-2-deoxycytidine treatment could not activate Lin28a transcription. In contrast, treatment with the histone deacetylase inhibitor Trichostatin A (TSA) induced Lin28a expression; this also increased the in vivo susceptibility of the Lin28a promoter DNA to nuclease digestion. We also observed an association between the expression status of Lin28a and the modification of the lysine 9 residue of histone H3 (H3K9): expression of Lin28a in undifferentiated embryonal carcinoma P19 cells and TSA-treated NIH/3T3 fibroblasts was consistently associated with the enrichment of acetylated H3K9 (H3K9Ac) and a reduction in dimethylated H3K9 (H3K9Me2) occupancy on the Lin28a promoter. The opposite occurred when Lin28a expression was silenced upon cellular differentiation or in the absence of chemical induction. The histone modification pattern also influenced the occupancy of RNA polymerase II on the Lin28a promoter. Our findings suggest that the dynamic change in H3K9Ac and H3K9Me2 occupancy is involved in the activation of Lin28a transcription by controlling the accessibility of the Lin28a promoter. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor promoting or suppressive effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly studied. We examined the expression pattern of miRNAs in the non-small cell lung cancer cell line H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono- and di-methylation of lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, we identified two miRNAs that showed consistent downregulation after BIX01294 treatment. Our results indicate histone H3 methylation regulates miRNA expression in lung cancer cells; this may provide additional insight for future chemical treatment of lung cancer.